Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Summary

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

Abstract

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.

Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease's progression and facilitate the development of potential anticancer drugs.

Introduction

Among all oncological disorders, lung cancer not only inflicts the highest life loss but also claims the second-highest number of new patients every year in the US¹. This devastating malignancy stands as a major obstacle in modern healthcare, urging for a deeper understanding of its intricate biology and more efficacious therapeutic modalities². Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer and tends to develop into solid tumors³. One of the foremost challenges in lung cancer is the dynamic and heterogeneous tumor microenvironment, which profoundly influences the cancer's ....

Protocol

The animal study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the University of the Pacific (Animal Protocols 19R10 and 22R10). Eight Male athymic nude mice aged 5-6 weeks, weighing 20-25 g, bred with the referenced Rodent Diet and housed under pathogen-free (SPF) conditions, were used for the present study. Cages, bedding, and drinking water were autoclaved and changed regularly. A schematic of tumor inoculation in mice is shown in Figure 1. .......

Discussion

The construction of A549-iRFP MCS is a straightforward and highly reproducible lab procedure and can be translated to MCS formation for multiple cell lines. The MCS generated with the aid of centrifugation and collagen exhibits a more integral and solid-tumor-like structure within 3-4 days. This method ensures the formation of robust spheroids that maintain their integral structure for extended periods, typically 2-3 weeks or even longer until small buddings begin to emerge. By employing centrifugation and collagen, we s.......

Materials

Name	Company	Catalog Number	Comments
100 µL Glass Syringe	Hamilton	Part/REF #80601
20 G Needle	Thermo Fisher Scientific Inc.	14 826D
96-well Ultra-Low-Attachment Spheroid Microplate	Corning	15-100-173
A549-iRFP	Imanis Life Sciences	CL082-STAN
AIN-93M Mature Rodent Diet	Research Diets, Inc.	D10012M
Athymic Nude Mouse	Charles River Laboratories, Inc.	Strain Code: 490; homozygous
BCA	Pierce	23227
Buprenorphine Hydrochloride	Patterson Veterinary	NDC Number: 42023-179-05
CellTiter-Glo 3D Cell Viability Assay	Promega	G9683
Collagen	Gibco	A1064401
DMEM	Corning	MT10013CV
Fetal Bovine Serum (FBS)	Cytiva HyClone	SH3039603
ImageJ	Open source tool (https://imagej.net/ij/)	N/A
Image Studio	LI-COR	Version 5.2
Isoflurane	Patterson Veterinary	NDC Number: 17033-0091-25
Ketamine	Patterson Veterinary	NDC Number: 50989-0161-06
Microscope	Keyence	Model number: BZ-X710
Matrigel	Corning	CB-40234
Odyssey Infrared Imaging 205 System	LI-COR	Model number: 9140
PBS	Corning	MT21040CV
Pearl Trilogy small animal imaging system	LI-COR	Model number: 9430
Penicillin-Streptomycin	Corning	MT30002CI
Puromycin	Thermo Fisher Scientific Inc.	AAJ67236XF
ReViSP software from MATLAB	Open source tool on Sourceforge (https://sourceforge.net/projects/revisp/)	N/A
Surgical Clips--AutoClip System	Fine Science Tools	12020-00
Xylazine	Patterson Veterinary	NDC Number: 61133-6017-01